Three walnut cultivars (superior line),‘Xiangling’,‘Lu W06-1’ and ‘Xiluo 3’,were used as experimental materials to study their cold-tolerance and the effect of different low temperatures and times on walnut’s osmolytes content and protein expression.Results showed that:(1)The ratio of xylem of ‘Lu W06-1’s 1-year-old twigs was the largest,and thickness of phloem of ‘Xiluo 3’ was significant larger than that of others.(2)With the temperature decreasing,relative electric conductivity (REC) increased gradually,and the REC of ‘Xiluo 3’ was almost larger than that of other two walnut cultivars (superior line) under every treatment.(3)The contents of soluble sugar (SS),free proline (FP) and soluble protein (SP) increasing firstly and then decreasing.However,the temperatures that increased to maximum value were still different.The SP contents of ‘Lu W06-1’ increased faster and larger,and its SS and FP contents were always larger than that of others under treatments.There were significant positive correlations between any two osmolytes of the three osmolytes,especially the correlation coefficient between SS and FP,which was 0.844.These indicated that their respond to cold stress were linked by a close relationship.(4)Electrophoretic protein patterns showed similar among different treatments,but there were significant accumulation existed in 6 protein bands of walnut branches when exposed to low temperature,which was from 38.9 kD to 87.9 kD.The cold tolerance abilities were ranked in the following order:‘Lu W06-1’>‘Xiangling’>‘Xiluo 3’.When exposed to low temperature,SP content of walnut branches accumulated rapidly,then SS and FP increased.In addition,more accumulated protein bands were detected in walnut branch which was more cold-tolerant.